The long-term goal of this project is to discover novel types of broad-spectrum antibiotics capable of effectively eradicating a wide range of potential biowarfare agents, as well as conventional pathogens. In this phase of the project, we will develop a high-throughput screen for a targeted discovery of prodrug antibiotics. The screen is aimed at solving the following problems that have impeded antimicrobial drug discovery: automatically eliminate a high background of generally- toxic molecules in compound library screening; obtain lead compounds with good penetration into Gram negative species, indicative of a broad spectrum of activity; and an ability to sterilize an infection by eliminating persister cells invulnerable to all current antibiotics. Prodrugs such as metronidazole convert inside the cell into a reactive molecule that hits multiple targets. In this regard, an activated prodrug acts like an antiseptic. Our preliminary data show that prodrugs can potentially completely sterilize a stationary state population of E.co//containing persisters. An ability to sterilize an infection can be critically important for treating immunocompromised individuals and multidrug tolerant biofilms that cause 65% of all infectious diseases in developed countries. An activated prodrug will bind covalently to its targets, creating an irreversible sink which will allow it to avoid efflux by MDR pumps of Gram negative species which is the main obstacle in developing broad-spectrum antibiotics. We will use E.'coli,.including the O157:H7 enterohemorrhagic strain that is a Category B waterborne agent in screen development. The process will include test strainconstruction- screening at the National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases facility in Boston and at an NIH Screening Center, and validation of hits to indicate their prodrug nature. This research will lead to the development of new antibiotics with a broad spectrum of action that will be effective against both bioweapons agents such as Y.pestis, F. tularensis and B. anthracis, and conventional pathogens. The uniquely novel property of these compounds will be their ability to completely sterilize an infection, preventing relapse of a disease and enabling effective treatment of immunodeficient individuals, including the elderly and the very young. PERFORMANCE,SITE(S) (organization, city, state) , Northeastern Mniversity, Boston, MA National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases, Boston, MA PHS 398 (Rev. 09/04) Page 2 Form Page 2 Principal Investigator/Program Director (Last, First, Middle): Lewis, Kim KEY PERSONNEL. See instructions. Usecontinuation pages as neededto provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first; Name eRA Commons User Name Organization Role on Project Kim Lewis k.lewis@neu.edu Northeastern University PI Gabriele Casadei Northeastern University PostdAssociate OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Richard E. Lee Univ of Memphis, TN Consultant Human Embryonic Stem Cells ^ No Q Yes If the proposed project Involves human embryonic stem cells, list below the registration number of the specific cell llne(s) from thefollowing list: http://stemcells.nih.qov/reqistrv/index.asp. Usecontinuation pages as needed. If a specific line cannot be referenced at this time, include a statement that one from the Registry will be used. Cell Line Disclosure PermissionStatement. Applicable to SBIR/STTR Only. See SBIR/STTR instructions. I I Yes I I No PHS 398 (Rev. 09/04) Page 3 Form Page 2-continued Number the following pages consecutively throughout the application. Do not use suffixes such as 4a, 45. Principal Investigator/Program Director (Last, First, Middle): Lewis, Kim The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description,